While a place for somatic gene therapy in the treatment of inherited single gene disorders is no longer disputed, the potential of gene therapy for the treatment of malignancies may not be readily apparent. Because most forms of cancer have been shown to be complex, multifactorial, and multigenic in nature, there are many conceptual and technical obstacles which must be overcome in order to approach this disease at the genetic level. Yet, it is the molecular nature of tumorigenesis, i.e., the activation of dominant oncogenes and/or the inactivation of tumor suppressor genes, that provides insight for such strategies in that these genetic events represent novel targets for molecular therapy. Already, genetic analysis is being used in diagnostic and prognostic predictions in certain malignancies (e.g., amplification of erb-B2 in breast and ovarian cancer; amplification of N-myc in neuroblastoma; and ras mutations in adenocarcinoma of the lung).
At present, there are two general strategies for gene therapy: gene augmentation and gene replacement. Gene augmentation or gene addition is simply the introduction of foreign genetic sequences into a cell. Usually this means the insertion of a normal copy of a particular gene into a cell expressing a mutant form of that gene. In many cases, the addition of functional genetic information has been used successfully to restore a genetic function in these defective cells. However, in other cases, the addition of a normal gene is not sufficient to repair the abnormality because this technique does not remove or correct the resident, nonfunctional mutant gene. Furthermore, the random insertion of foreign sequences into nonspecific sites of the genome may result in mutagenic events such as insertional inactivation of genes necessary for the viability of that cell, or uncontrolled regulation of the transgene and/or flanking chromosomal sequences. In these instances it will be necessary to modify specific gene sequences by targeted gene replacement, i.e., site-specific recombination of foreign DNA into targeted genomic sequences. Each of these approaches have applications in gene therapy for cancer.
The immune system has demonstrated the potential to play a protective role in cancer. However, the vast majority of malignancies arise in immunocompetent hosts. Although cellular activity is normally regulated by the various protein kinases, growth factors, growth factor receptors, and DNA binding proteins encoded by proto-oncogenes, together with genes that can suppress malignant transformation, such as the retinoblastoma and p53 genes, if one or more of these genes is or becomes defective, it can result in a clone with an abnormal pattern of growth control. The fact that such a clone grows uncontrolled in an individual, indicates that the immune system has either failed to recognize tumor-specific antigens or has failed to effectively respond. Transgenic immunotherapy, an important arm of somatic gene therapy for cancer, aims at strengthening the immune surveillance of the body.
It is known that the presence of cytokines at the site of a tumor can drastically alter tumor/host relations. In some cases a highly destructive and specific response to otherwise nonimmunogenic tumors can be elicited by the insertion of genes encoding cytokines (e.g., interleukin-2, interleukin-4, interferon-γ, and tumor necrosis factor) into tumor cells which are then used as “tumor vaccines.” Anti-tumor responses can also be enhanced by the transfection of these genes into cytotoxic lymphocytes or macrophages. Autologous tumor-infiltrating lymphocytes have been used successfully in such genetic immunomodulation studies because of their inherent specificity for the tumor, and their ability to home back to the tumor site when reinfused into the patient. This approach has been termed “adoptive immunotherapy.” It is also possible to protect normal tissue by stably transfecting normal bone marrow cells with cytokine genes prior to chemotherapy, thereby achieving a more continuous effect while obviating the need to infuse these drugs which have short half-lives and produce systemic side effects when delivered intravenously.
Despite the widespread use of chemotherapeutic agents for the treatment of solid tumors, efficacy has been restricted by their toxicity to normal cells. Transfection of normal stem cells with transgenes conferring resistance to these agents would result in cytotoxic drug-resistant cells and allow the administration of more therapeutically significant doses. Another type of gene therapy that is gaining momentum and which is now in clinical trails is the use of “informational drugs.” Antisense oligonucleotides, small synthetic nuclease-resistant nucleotide sequences complementary to specific RNA sequences, are perhaps the best known example of this. By specifically binding and thereby inhibiting transcription and/or translation of a single oncogene, it may be possible to reverse clinical symptoms.
It is well established that T lymphocytes recognize two different types of antigens, one being peptides derived from conventional protein antigens and the other being superantigens. The classical model for superantigen activity suggests that the superantigens react in some ways like conventional antigens but exhibit critical differences in others (Johnson et al., 1992). Before a T helper cell can recognize conventional protein antigens, these proteins must first undergo processing by macrophages or other antigen presenting cells (APC). APCs then display the peptide on the cell surface in combination with MHC. Unlike typical antigens, however, superantigens bind MHC directly without uptake and processing by APCs (Johnson et al., 1992).
Unlike conventional antigens, where recognition by T cells involves both variable elements of the α and β chains of TCR, superantigen recognition depends primarily on the TCR Vβ region. Also, unlike ordinary antigens, superantigens bind to specific Vβ segments of TCR which are outside of the normal antigen-binding groove. This binding occurs regardless of the remaining structure of the TCR. These interactions lead to strong Vβ-specific T cell activation (Dellabona et al., 1990, and Rust et al., 1990). Furthermore, because the number of different types of Vβ segments is small compared with the number of α, β receptors, many more T cells are capable of recognizing a particular superantigen than are able to identify a specific antigen (Herman et al., 1991).
The classical model of superantigen activity has since been revised. New observations suggest that there are additional interactions between the TCR and MHC molecules during superantigen engagement and this can have a significant impact on superantigen specificity and function (Webb and Gascoigne, 1994). For example, some studies show that it is not only Vβ, but also the α chain of the TCR which plays a role in the recognition of superantigens (Karp et al., 1990, and Panina et al., 1992). Furthermore, the MHC molecule does not function as an inert platform for superantigen presentation, but plays a role in T cell activation by superantigens. Some studies indicate that individual T cell clones are able to distinguish superantigens presented on MHC molecules with different specificity. Some murine T cell hybridomas can recognize superantigens in context of two different MHC molecules (Mollick et al., 1991; Hartwig and Fleischer, 1993). The revised model assumes that the molecular mechanism of T cell stimulation is probably a multivalent cross-linking of the TCR with MHC molecules. Additional adhesion molecules such as CD 2, LFA-1 or CD 28, may play a role during T cell stimulation (Fleischer and Hartwig, 1992, and Fraser et al., 1992). Another interesting observation suggests that in spite of the requirement for MHC class II molecules in T cell stimulation, there is evidence that superantigens interact with TCRs directly, in the absence of class II molecules. Binding to MHC II is not a prerequisite for T cell activation as superantigen-mediated cytotoxicity has been found against several class II-negative target cells. However, the interaction of the superantigen with TCR is apparently of low avidity and is usually insufficient to generate a full response (Dohlsten et al., 1991, Herman et al., 1991, and Avery et al., 1994).
Two general categories of superantigens have been described. The soluble exotoxins produced by gram-positive bacteria such as Staphylococcus aureus typify bacterially-derived superantigens and are well known for their ability to cause food poisoning and symptoms of shock. Viral superantigens have also been described. In mice, prototypical viral superantigens are encoded by endogenous mouse mammary tumor viruses (MMTVs). These viral superantigens include M1s antigens and are strongly immunogenic for murine T cells.
Because of their ability to stimulate strong T-cell responses in vivo, superantigens have elicited wide interest. M-like proteins of group A streptococci act as a key virulence factor on the bacterial surface. M protein is defined by its antiphagocytic function, whereas M-like proteins, while structurally related to M protein, lack an established antiphagocytic function. The emmL 55 gene, derived from the M stereotype 55 group A streptococci isolate A928, has an amino acid sequence typical of M-like proteins (Boyle et al., 1994, and Boyle et al., 1995).
Bacterial superantigens, when covalently linked to mAbs specific to the cell surface molecules of malignant, MHC II− target cells, can direct lysis of these cells (Dohlsten et al. 1991). It has been demonstrated that conjugation between the superantigen staphylococcal enterotoxin-A (SEA), and mAbs recognizing human colon cancer enabled T cells to lyse colon carcinoma cells in vitro. The use of Staphylococcal enterotoxins has been contemplated for cancer vaccines (WO 95/00178).
Immunotherapeutic modalities for the treatment of oncological disorders have been described by a number of scientific investigators. The immunological rejection of tumors has been shown in response to the transfection of tumor cells by such antigens expressed by MHC genes (Hock et al., 1996) and Mycobacterium (Menard et al., 1995). Research into immunotherapy for oncological diseases using MHC antigens and the delivery thereof has been described (EP 569678; WO 95/13092). Other antigens, bacterial and viral, have also been used in combination with cytokine or other immunomodulator gene expression and delivered by means of adenovirus, retrovirus or plasmid vectors (WO 94/21808; WO 96/29093).